Mass spectrometry has become an important tool for understanding how molecules function in sample systems such as biological tissue. Large R&D investments have been dedicated to the technological progress of mass spectrometers. To add value to these investments, small businesses have developed add-on ionization sources that extend the capabilities of the instrument. These third party ionization sources have enabled researchers to create mass spectrometry images (spatial molecular mass maps) from tissue samples, improve sensitivity and directly analyze samples without sample preparation in real time. Matrix-assisted laser desorption and ionization (MALDI) has become the ionization source of choice for many of these third-party ionization sources especially for MS imaging (MSI) because a laser beam can be focused to a very small spot size to increase spatial resolution. Typically, the laser operates in the UV and desorbs/ionizes the sample in the presence of a light absorbing matrix. The need for matrix addition is a limiting factor because the even distribution of the matrix across the sample surface can be time consuming, expensive, irreproducible and require extensive expertise. It has been demonstrated that mid-IR lasers can successfully ionized biomolecules in tissue by using water as an endogenous matrix. Direct mid-IR ionization has successfully been used in atmospheric pressure (AP) MALDI sources as well as an efficient desorber for secondary ionization by Electrospray Ionization (ESI) and Direct Analysis in Real Time (DART) sources. Optical parametric oscillators (OPO) have become the laser technology of choice for generating mid-IR light. Focusing the laser light to very small spot sizes has been a problem due to the high divergence of OPO beam. Cost and size of the OPOs prevent the technology from being widely adopted. This grant proposal aims to develop a mid-IR OPO with a divergence close as possible to the diffraction limit in Phase I. The OPO will be tested by creating highly resolved MS images of biological tissue samples will little to no sample preparation or pretreatment. Phase II will address cost and size issues. PUBLIC HEALTH RELEVANCE: Funding of this grant proposal will provide the resources to develop a better tool for creating a "biological image" of tissues and cells. The image will show the location of important molecules, such as metabolites and proteins, during different stages of biological development. This information will help scientists understand how life functions in its native form or when a foreign substance such as a drug is introduced into the system.